DESCRIPITON (provided by applicant): Humans and animals cannot synthesize vitamin K (K). K dependent, hypoprothrombia is an important clinical problem under a number of conditions. The K requirement in human and animal nutrition is met by green plants and intestinal bacteria. Because K plays an important role in human well being, it is important to understand its biosynthesis and regulation. The vitamin K biosynthetic pathway may be summarized as follows: chorismate -menF--> isochorismate --menD--> 2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1 -carboxylate (SHCHC) ---menC---->o-succinylbenzoic acid (OSB)-menE-> o-succinylbenzoyl-CoA (OSB-CoA) --menB-->{ 1, 4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA)}---menH---> 1, 4-dihydroxy-2-naphthoic acid (DHNA) ---menA---> demethylmenaquinone (DMK) --- ubiE --> menaquinone (MK). Seven ORFs (six of which have been shown to be menaquinone biosynthetic genes) lie in a cluster in the order orf1O1, menF, menD, menH, menB, menC, and menE. Transcription of all the genes are initiated from a single strong promoter site, 26 bp 5' to orfiOl was detected under anaerobic conditions. The menA gene is located at 89 min on the chromosome and is linked to an unidentified orf (orf161). In this study, we propose 1) to determine the role of orf101; 2) to study the regulation of the men operon using lacZ fusions; 3) to determine the structure of a proposed intermediate (DHNA-CoA); 4) to identify the methyltranferase involved in the DMK-->MK conversion, and 5) to characterize the active site mutants of OSB synthase, whose three dimensional structure was recently solved by us.